Method for extracting urokinase

ABSTRACT

ACRYLONITRILE MATERIAL IS USED TO ADSORB UROKINASE FROM A LIQUID, AND SAID UROKINASE IS SUBSEQUENTLY ELUTED FROM SAID MATERIAL.

Patented Mar. 27, 1973 Int. C1. C05,; 7/026 US. Cl. 195-66 B 4 Claims ABSTRACT OF THE DISCLOSURE Acrylonitrile material is used to adsorb urokinase from a liquid, and said urokinase is subsequently eluted from said material.

SUMMARY OF THE INVENTION The present invention relates to a method for extracting a condensed, purified urokinase from human urine on an industrial scale. Urokinase is known to be an enzyme occurring in small quantities in the human urine and found effective for treating various forms of thrombosis, because, when intravenously injected into the human body,

it can dissolve pathologically formed fibrin or fibrinous coagulations clogging the blood vessels.

For the extraction of urokinase from human urine, various available media are known, such as: heavy metals, barium sulfate, silicic acid and its salts, and various ion exchangers.

Having sought for a method more eflective than the known methods, the present inventors discovered one which is more eflicient than any of the known methods and simple to carry out.

The method according to the present invention is characterized by the step of adsorbing the urokinase in human urine on a material never utilized for extraction of urokinase, that is to say, an arcylonitrile synthetic fiber or its fabrics such as Bonnel (trade name of product by Mitsubishi Bonnel), Exlan (trade name of product by Japan Exlan) or Kashimiron (trade name of product by Asahi Kasei). The following isa description of the fiber but it applies also for its fabrics.

The acrylonitrile synthetic fiber to be used in this invention is so cheap and easily accessible that it can be thrown away after use, and it needs no pretreatment before use. If, however, it is desirable to remove minor stains from the fiber, the purpose is simply attained by preliminarily dipping the fiber in water and stirring it therein. When air bubbles entrapped among fibers are to be eliminated before contacting the fibers with urine, a similar treatment will help.

Since the fibers are sufficiently effective in small quantities and the urokinase adsorbed thereon can be easily separated, our new method is economically superior to any conventional method.

Another feature of our new method resides in the fact that manpower for the collection of urine can be saved or even made unnecessary.

Specifically, the enormous manpower required for collection of urine can be replaced by the simple collection of small, light acrylonitrile fibers overlying the urinal or filling a portion of the drain pipe connected to the bottom of the urinal. Thus the invented method will be highly useful for industrial extraction of urokinase.

The process according to our new method is roughly as follows: Human urine is brought into contact with acrylonitrile synthetic fibers, so as to adsorb urokinase from the urine into these fibers, which are picked out to be washed with water to remove the residual urine. Elution is then made using an alkaline solution. Since the urine composition is heavily influenced by' the intake of food or water, the movement, time or climate, it is difiicult to define a normal pH value for the urine, but it is supposed to range generally between 4.8 and 7.5 (Hidenobu MAJIMA: Physiology published by Bunkodo, 1968). In this pH range of 4.8-7.5 urokinase can be adsorbed on acrylonitrile synthetic fibers, but for the sake of a higher yield the pH range of 5.5-6.5 would be preferable. When, for example, a 4% aqueous solution of ammonia is employed for the elution of adsorbed urokinase, an elution rate of 94% will be attained.

The contact between the urine and the acrylonitrile synthetic fibers is rendered more effective by following the above procedure after treating the urine at room temperature with its pH value adjusted to 7.5-9.5 or preferably to 8.5, and thereby removing the resultant sediments.

The urokinase-containing solution thus obtained is condensed or refined into a preparation suitable for medical purposes. It is well known that, for the purpose of condensation, the following methods are available: salting-o ut by means of ammonium sulfate, sedimentation by means of an organic solvent, Diafio (trade name of product by U.S. Amicon). For the purpose of refining, various ion exchangers may be utilized, but a high-purity urokinase will be obtained by another contact with acrylonitrile synthetic fibers.

Next, the selective adsorption of urokinase on acrylonitrile synthetic fibers will be specifically demonstrated. Table 1 gives the yields of urokinase from various fibers when 2 g. of fibers was contacted with 3 l. of urine having a pH value of 6.0. The fibers were washed with water and the unokinase was then eluted using a 4% aqueous solution of ammonia.

TABLE 1.-YIELDS OF UROKINASE Main compo- Yield Fibers sitlou (percent) Absorbent cotton Cellulose 8 Tevrronfl (trade name of product by Vinylchloride 15 Terjm, vinyl chloride synthetic fiber.)

Bonnel Acrylonitrile.... 89

Table 1 demonstrates the superiority of our new method with respect to the yield of urokinase. Table 2 is a comparison between conventional methods and our new method.

TABLE 2.COMPARISON BETWEEN CONVENTIONAL AND INVENTED METHODS It is clear from Table 2 that the invented method gives a higher yield of urokinase than conventional methods. Meanwhile it should be noted that the so-called absorbents hitherto known adsorb not only urokinase but also great masses of impurities, so that the purity of the product of elution is low, and the value of these adsorbents lies simply in the condensation of urokinase out of urine.

It is clear from Tables 1 and 2 that the invented method is an excellent one characterized not only by the possibility of elution in a condensed state but also b" high selectivity in urokinase adsorption.

Units of urokinase have been determined by the fibrin plate method advocated by Ploug et a1 [1. Ploug: Biochem. Biophys. Acta, vol. 24, p. 278 (1957)].

The following is a description of the present invention, with some examples of its execution. This will make the present invention more clearly understandable, but the invention is not limited to the cited examples only.

EXAMPLE 1 A large volume of fresh urine collected from many healthy people was adjusted to pH 8.5 under agitation, using sodium hydroxide. The resulting sediment was filtered away or eliminated by centrifugal separation. Then, under violent agitation, 6 N hydrochloric acid was gradually added for adjustment of pH 5.5. This was immediately followed by the addition of Bonnel at a rate of 70 g. per 100 l. of urine and by violent agitation. The Bonnel" which had thus been brought into contact with urine was gathered in a large funnel and washed with water until the wash water turned colorless and clear. Then by compression the wash water was removed. Next using 2 l. of a 4% aqueous solution of ammonia, the urokinase adsorbed by the fibers was separated by elution. Ammonium sulfate was added to the eluent under agitation to 60% saturation. After agitation had been continued further for one hour, the precipitate was collected for 30 minutes of centrifugal separation at 20,000 G, dissolved in 500 ml. of water, and then subjected to overnight dialysis, using 100 times as much water. The solution thus obtained contained about 85% of the urokinase in the original urine, its specific activity being about 4,000 units/mg.

EXAMPLE 2 Under vigorous agitation, 6 N hydrochloric acid was gradually added to a crude urokinase solution obtained as in Example 1 for adjustment to pH 5.5. Immediately thereafter, Bonnel was added to the crude urokinase solution at a rate of 100 g. per 1 l. of said solution and thoroughly stirred. Subsequent treatment was the same as in Example 1 and elution was carried out using 1 l. of a 4% aqueous solution of ammonia. Thereafter the process was the same as in Example 1.

The solution finally obtained contained about 80% of urokinase in the crude urokinase solution, its specific activity being over 13,000 units/mg.

EXAMPLE 3 100 g. of kaolin was added to 100 l. of fresh urine from which sediment had been removed by filtration, and then the mixture was thoroughly stirred. The kaolin was collected by centrifugal separation and washed until the washing turned colorless and clear. Next, the kaolin-adsorbed urokinase was separated by elution using 3 l. of a 4% aqueous solution of ammonia and then subjected to overnight dialysis. The crude urokinase solution thus yielded was adjusted to pH 5.5 using 6 N hydrochloric acid. g. of Exlan (trade name of product of Japan Exlan) textile pieces was then added and well agitated. Thereafter the same process as in Example 1 was carried out. Namely, elution was performed using 1 l. of a 4% aqueous solution of ammonia. Thereafter the process was the same as in Example 1.

The solution thus obtained contained about of urokinase in the crude solution, or about 40% of it in the original urine, its specific activity being over 10,000 units/mg.

What is claimed is:

1. A method for extracting urokinase from a liquid containing said urokinase, which method comprises the steps of bringing a material comprising synthetic acrylonitrile fibers into contact with said liquid until a substantial proportion of said urokinase has been adsorbed by said material, and then separating the adsorbed urokinase from said material by elution.

2. The method claimed in claim 1 in which said urokinase is eluted with an alkaline solution.

3. The method claimed in claim 1 in which the pH value of said liquid is adjusted to from 5.5 to 6.5 before said urokinase is adsorbed.

4. The method claimed in claim 1 in which said fibers are in fabric form.

References Cited UNITED STATES PATENTS 3,542,646 11/1970 Aokietal -66B 3,650,903 3/1972 Sloane 195-66 B LIONEL M. SHAPIRO, Primary Examiner 

